title: Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi.
abstract: Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.
authors.affiliation: Department of Pathology, University of California, San Francisco, California 94143, USA. jcengel@itsa.ucsf.edu
authors.forename: J C
authors.initials: JC
authors.lastname: Engel
authors.forename: P S
authors.initials: PS
authors.lastname: Doyle
authors.forename: J
authors.initials: J
authors.lastname: Palmer
authors.forename: I
authors.initials: I
authors.lastname: Hsieh
authors.forename: D F
authors.initials: DF
authors.lastname: Bainton
authors.forename: J H
authors.initials: JH
authors.lastname: McKerrow
mesh_terms: D000818:Animals; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D008247:Lysosomes / Q000187:drug effects; D015800:Protozoan Proteins / Q000187:drug effects*; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000648:ultrastructure
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi